Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level.
Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level.
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DOI：
10.1016/j.jphotobiol.2008.12.010
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发表时间：
2009-04-02
影响因子：
5.4
通讯作者：
Heikal, Ahmed A.
中科院分区：
文献类型：
--
作者：
Yu, Qianru;Heikal, Ahmed A.
还原型烟酰胺腺嘌呤二核苷酸（NADH）是细胞氧化磷酸化和糖酵解途径中的主要电子供体。因此，最近出现了采用内源性NADH荧光作为一系列细胞过程的天然探针的复苏，包括细胞凋亡，癌症病理学和酶动力学。在这里，我们报告了乳腺癌（Hs 578 T）和正常（Hs 578 Bst）细胞中内源性NADH的双光子荧光寿命和偏振成像，用于定量分析浓度和构象（即，游离与酶结合的比率）。细胞内NADH的双光子荧光寿命成像表明对细胞病理学和使用氰化钾（KCN）抑制呼吸链活性的敏感性。使用新开发的非侵入性测定法，我们估计癌细胞中的平均NADH浓度（168 ± 49 μM）比乳腺正常细胞（99 ± 37 μM）高约1.8倍。这种分析表明，在正常乳腺细胞中的能量代谢和氧化还原反应的变化后，使用KCN的呼吸链活性的抑制。此外，细胞自发荧光的时间分辨相关各向异性指示正常乳腺细胞中细胞内NADH的游离构象（0.18 ± 0.08）和酶结合构象（0.82 ± 0.08）的群体分数。这些分数与乳腺癌细胞中的分数存在统计学差异（游离：0.25 ± 0.08;结合：0.75 ± 0.08）。溶液中NADH与线粒体苹果酸脱氢酶和乳酸脱氢酶结合动力学的比较研究模拟了我们在活细胞中的发现。这些定量研究证明了细胞内NADH动力学（而不是强度）成像用于探测与神经退行性疾病、癌症、糖尿病和衰老相关的线粒体异常的潜力。我们的方法也适用于活细胞中的其他代谢和信号传导途径，而不需要像常规生化测定那样破坏细胞。
Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed noninvasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ~ 1.8 fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.
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影响因子：
3.8
作者：
Chia, Thomas H.;Williamson, Anne;Levene, Michael J.
通讯作者：
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DOI：
10.1016/j.jphotobiol.2005.04.001
发表时间：
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